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Human Molecular Genetics Advance Access originally published online on November 2, 2007
Human Molecular Genetics 2008 17(3):419-430; doi:10.1093/hmg/ddm319
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Germline mutations of MEK in cardio-facio-cutaneous syndrome are sensitive to MEK and RAF inhibition: implications for therapeutic options

Thanaset Senawong1,2, Janyaporn Phuchareon1,2, Osamu Ohara4, Frank McCormick2, Katherine A. Rauen2,3 and Osamu Tetsu1,2,*

1 Department of Pathology, School of Medicine 2 UCSF Comprehensive Cancer Center and Cancer Research Institute 3 Department of Pediatrics, School of Medicine, University of California, San Francisco, CA 94143-0128, USA 4 Department of Human Genome Research, Kazusa DNA Research Institute, Kisarazu, Chiba 292-0818, Japan

* To whom correspondence should be addressed at: 2340 Sutter Street N111, UCSF Mt Zion Cancer Research Building, San Francisco, CA 94143-0128, USA. Tel: +1 4155140870; Fax: +1 4155023179; Email: tetsu{at}cc.ucsf.edu

Received September 9, 2007; Accepted October 29, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Cardio-facio-cutaneous (CFC) syndrome is a sporadic developmental disorder characterized by distinctive craniofacial features, heart defects, mental retardation and ectodermal abnormalities. We recently reported missense germline mutations in the genes MEK1 and MEK2 in patients with CFC. These mutations, including F53S and Y130C MEK1, and F57C MEK2, are the first naturally occurring mutations to be identified in these genes. This study reports data concerning the biochemical functions of the novel mutants, as well as the roles of these MEK genes in the MAPK signaling cascade. Our CFC MEK variants cannot induce ERK unless they are phosphorylated by RAF at two key serine residues in the regulatory loop. When we replaced the serine residues with alanines, ERK phosphorylation was significantly reduced in the presence of RAF. We did find that F57C MEK2 activation was less dependent on RAF signaling than the other mutants. This difference results in F57C MEK2 being resistant to the selective RAF inhibitor SB-590885. All three mutants are sensitive to the MEK inhibitor U0126. The majority of CFC cases result from mutations in B-RAF. A recent report indicates the possibility that cancer cells with activated B-RAF have enhanced, selective sensitivity to MEK inhibitors. Thus, regardless of mutations identified in an individual with CFC, MEK inhibition is a potential therapeutic approach for this population.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Mitogen-activated protein kinases (MAPKs) are serine or threonine protein kinases that respond to stimulation from extracellular growth factors through specific cell surface receptors. They are part of a major signaling cascade, which is activated in the sequence of RAS, RAF (also known as MAPKKK), MEK1/2 (MAPKK) and ERK1/2 (classical MAPK) (1–3). The activation of this pathway regulates a variety of cellular processes, including proliferation and differentiation (1,4).

Gain-of-function mutations in RAS or RAF are frequently found in human neoplasms, where they contribute to oncogenesis and tumor progression (2,5). Recently, gain-of-function mutations in this cascade have been found in genetic syndromes such as Costello syndrome (CS) and cardio-facio-cutaneous (CFC) syndrome (6–8). CS and CFC are developmental disorders with overlapping features. These include craniofacial dysmorphia, hair and skin abnormalities, cardiac anomalies and postnatal growth deficiency, although each syndrome is unique and has its own clinical course (4,6–8). Patients with CS have an elevated incidence of malignancy (6). In contrast, it is yet unclear if CFC individuals are at an increased risk for cancer during their childhood or in their later lives. This may be a reflection of the limited number of patients with this disease (4). Recent report demonstrated that three individuals from CFC have developed neoplasms in different tissues, resulting in rhabdomyosarcoma, hepatoblastoma and acute lymphoblastic leukemia (9–11). CS is associated with mutations in H-RAS (6). We recently identified that CFC is caused by mutations in B-RAF, MEK1 and MEK2 (7,12). The mutations we found in MEK1 and MEK2 are the first naturally occurring ones identified in these genes (7). Elucidating their biochemical functions is important for understanding MEK's role in the Ras/MAPK signaling pathway in these genetic syndromes and cancer. It could also provide information for approaches to therapy in CFC and malignancies. MEK1 and MEK2 are promising targets for small molecule inhibitors that block the MAPK signaling pathway (2,3). This study demonstrates potential target sites within these molecules in CFC.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
{Delta}N3, F53S and Y130C MEK1 mutants require RAF signals to activate ERK1/2
Missense germline mutations of MEK1 and MEK2 in patients with CFC syndrome include the following: in MEK1, a serine substitution for phenylalanine 53 and a cysteine substitution for tyrosine 130 (F53S and Y130C), and in MEK2, a cysteine substitution for phenylalanine 57 (F57C; 7). In cultured cells, these mutants can activate endogenous ERK/MAPK more efficiently than wild-type (WT) MEK1 and MEK2. We believe that MEK1 and MEK2 mutants in CFC syndrome function actively in the Ras/MAPK signaling cascade. However, it is not known whether they can activate their downstream effector ERK1/2 without prior upstream signaling from growth factors (13). We addressed this question and found that the F53S and Y130C mutants stimulated ERK/MAPK in the presence of epidermal growth factor (EGF), but not in its absence.

We introduced expression vectors containing F53S, Y130C and F57C mutant MEK1/2 into human embryonic kidney (HEK) 293 cells and assessed endogenous ERK1/2 activation in the presence or absence of EGF. We compared the activities of these CFC mutants with other variants of MEK1/2. They include K97M (methionine substitution for the catalytic lysine 97 in MEK1) and K101M (a catalytic lysine change at position 101 in MEK2), {Delta}N3 (deletion of the N-terminal {alpha}-helix auto-inactivation domain in amino acids 32–51 of MEK1; 13), S218D+S222D (substitution of aspartic acids for serines 218 and 222 in the regulatory loop of MEK1), S222D+S226D (same in serines 222 and 226 in the regulatory loop of MEK2), S218A+S222A (substitution of alanines for serines 218 and 222 of MEK1) and S222A+S226A (same at serines 222 and 226 of MEK2). These forms of MEK1 and MEK2 are catalytically inactive (K97M MEK1 and K101M MEK2), active ({Delta}N3 MEK1), constitutively active (S218D+S222D MEK1 and S222D+S226D MEK2) or inactive (S218A+S222A MEK1 and S222A+S226A MEK2; 13,14).

Cells were serum-starved for 24 h and then cultured with or without EGF for 5 min prior to harvesting. ERK1/2 activity was determined by the Western blot analysis. We observed constitutive activation of ERK/MAPK in cells transfected with S218D+S222D MEK1 (Fig. 1A, lanes 9 and 10) and S222D+S226D or F57C MEK2 (Fig. 1B, lanes 7, 8, 11 and 12). These mutants stimulated ERK/MAPK activities in the absence of EGF. In contrast, we observed that EGF significantly increased the activities of ERK/MAPK in the samples transfected with F53S or Y130C MEK1 (Fig. 1A, lanes 14 and 16). We did not see significant induction of ERK/MAPK activities in the other samples (Fig. 1A and B). Similar profiles were obtained when the experiments were performed in COS-7 cells (data not shown). These observations demonstrate that depleting mitogens from cell cultures impaired F53S and Y130C MEK1 phosphorylation ERK1/2 (Fig. 1A, lanes 13 and 15; 7).


Figure 1
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  		Figure 1. Depleting mitogen signals from cell cultures impairs the abilities of the MEK1 mutants {Delta}N3, F53S and Y130C to phosphorylate ERK1/2. F57C MEK2 but not F53S and Y130C MEK1 can activate ERK1/2 without prior upstream signaling from EGF. HEK293 cells were plated into six-well plates. Twenty-four hours later, subconfluent cells were transfected with 2 µg each of expression vectors containing the various HA epitope-tagged MEK1 or MEK2 mutants. After an overnight incubation, the cells were serum-starved for 24 h with medium containing 0.5% FBS hours. Cultures were incubated for 5 min in the presence (+) or absence (–) of 10 ng/ml EGF prior to harvesting. Western blot analysis was performed using antibodies against HA for MEK1/2 variants, phosphorylation-specific p44 ERK1 and p42 ERK2 (P-ERK), and total p44 ERK1 and p42 ERK2 (Total ERK). (A) The following vectors were independently transfected: pcDNA3.1 (empty), WT, K97M (catalytically inactive), {Delta}N3 (active), S218D+S222D (constitutively active), S218A+S222A (constitutively inactive) MEK1, F53S and Y130C MEK1 (found in CFC). (B) The following were independently transfected: pcDNA3.1 (empty), WT, K101M (catalytically inactive), S222D+S226D (constitutively active) and S222A+S226A (constitutively inactive) MEK1 and F57C MEK2 (found in CFC).

 


Figure 2
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  		Figure 2. The MEK1 mutants {Delta}N3, F53S and Y130C require RAF signals to activate ERK1/2. HEK293 cells were co-transfected with 1.5 µg of various forms of MEK1 expression vectors and 0.5 µg of pcDNA3.1 (A) or an Myc epitope-tagged WT B-RAF DNA plasmid (B). Cells were serum-starved for 24 h in medium containing 0.5% FBS, harvested and western blot analysis was performed. Ectopically expressed WT B-RAF was detected with an antibody to Myc.

 
We next determined whether the F53S and Y130C mutants require RAF signaling to activate ERK. We studied this question because RAF is the sole activator of MEK1/2 in response to growth factors signaling through RAS (1). We transfected the various MEK1 expression vectors into HEK293 cells together with an empty vector (Fig. 2A) or a WT Myc epitope-tagged B-RAF DNA plasmid (Fig. 2B). Cells were serum-starved for 24 h, harvested and western blot analysis was performed. As expected, adding WT B-RAF dramatically restored the abilities of {Delta}N3, F53S or Y130C MEK1 to activate ERK1/2 (Fig. 2B, lanes 4, 7 and 8). Comparable data were obtained when we repeated these experiments in COS-7 cells (data not shown). These observations suggest that the {Delta}N3, F53S and Y130C MEK1 mutants require RAF to activate the MAPK pathway.

{Delta}N3, F53S and Y130C MEK1 require phosphorylation of serine residues 218 and 222 to activate ERK1/2
All three RAF family members phosphorylate two conserved key serine (Ser) residues in the regulatory loop of MEK1 and MEK2 (13–15). These sites are Ser218 and Ser222 (MEK1) and Ser222 and Ser226 (MEK2). Phosphorylation is necessary for activating MEK1/2 in the MAPK signaling cascade. We tested whether B-RAF phosphorylates {Delta}N3, F53S or Y130C at Ser218 and Ser222 and then activates ERK1 and ERK2 downstream.

We transfected different forms of MEK1/2 expression vectors into HEK293 cells, with or without a WT B-RAF plasmid (Fig. 3A and B). Cells were serum-starved for 24 h and then harvested. Western blot analysis was performed using phosphorylation specific-antibodies against MEK and ERK. We observed that {Delta}N3, F53S or Y130C activated ERK in the presence of WT B-RAF and that they were phosphorylated at Ser218 and Ser222 (Fig. 3A). In contrast, adding WT B-RAF caused little enhancement of MEK1 and ERK 1/2 phosphorylation in the other samples. Figure 3B shows that WT B-RAF induced phosphorylation of F57C MEK2 at Ser222 and Ser226. However, this modification did not cause further phosphorylation of ERK1/2. Ectopically expressed WT MEK2 was also phosphorylated in the presence of WT B-RAF, although to a lesser extent. This caused slight activation of endogenous ERK/MAPK. Similar data were obtained in COS-7 cells (data not shown).


Figure 3
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  		Figure 3. {Delta}N3, F53S and Y130C MEK1 mutants must be phosphorylated at serine residues 218 and 222 to activate ERK1/2. Various MEK1/2 expression vectors were independently transfected into HEK293 cells with (+) or without (–) an Myc epitope-tagged WT B-RAF DNA plasmid. Cells were serum-starved for 24 h in medium containing 0.5% FBS and then harvested. Western blot analysis was performed using antibodies against HA for MEK1, Myc for B-RAF, S218/S222 phosphorylation-specific MEK1 (P-MEK1) or S222/S226 P-MEK2, P-ERK and total ERK. (A) {Delta}N3, F53S and Y130C MEK1 mutants activated ERK when they were phosphorylated at Ser218 and Ser222 in the presence of WT B-RAF. (B) WT B-RAF induced phosphorylation of F57C MEK2 at Ser222 and Ser226 without further enhancement of ERK1/2 phosphorylation.

 
These observations suggest that (i) {Delta}N3, F53S and Y130C MEK1 mutants cannot activate ERK1/2 unless they are phosphorylated at Ser218 and Ser222 by upstream signaling from RAF and (ii) F57C MEK2 could be a constitutively active mutant such as S218D+S222D MEK1 and S222D+S226D MEK2.

To test the first possibility, we introduced further mutations in the {Delta}N3, F53S and Y130C mutants. We replaced the serine residues at positions 218 and 222 with alanines. This action reduced ERK phosphorylation in the presence of WT B-RAF (Fig. 4). We concluded that {Delta}N3, F53S and Y130C MEK1 mutants require activation by RAF for ERK induction.

Phenylalanine 57 of MEK2 is a unique site that harbors gain-of-function mutations
MEK1 and MEK2 are dual-specificity kinases that activate ERK1 and ERK2 (1,3). They share 79% amino acid identity and have similarity in their three-dimensional structures (16,17). These facts suggest that substitutions in the same positions in each enzyme may have similar functional consequences (7). However, Figures 1 and  2 show that mutants with mutations in equivalent residues—F53S MEK1 and F57C MEK2—require different levels of RAF signaling for activating ERK/MAPK. The discrepancy is likely due to differences in MEK1 and MEK2's requirements for activating the MAPK signaling cascade. It may mean that MEK mutants could be constitutively active forms only when the mutations are introduced into equivalent positions in MEK2.

To test this possibility, we introduced identical amino acid changes into equivalent positions of each enzyme (Fig. 5A and B). They are F57S MEK2 (lane 9) for F53S MEK1 (lane 8), F53C MEK1 (lane10) for F57C MEK2 (lane 11) and Y134C MEK2 (lane 13) for Y130C MEK1 (lane 12). We then compared each variant's activity on endogenous ERK1/2 activation. HEK293 cells were transfected with MEK expression vectors and serum-starved for 24 h before harvesting and western blot analysis (Fig. 5A). F57S MEK2 was equivalent to MEK1 F53S. ERK activity in F57S MEK2 was dramatically increased, compared with the MEK1 mutant (Fig. 5A, lane 9). Conversely, F53C MEK1, which was a mutation equivalent to F57C MEK2, rendered MEK1 inactive (Fig. 5A, lane 10). We did not see any changes in ERK activation caused by Y134C MEK2 (equivalent change to Y130C MEK1; Fig. 5A, lane 13).


Figure 4
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  		Figure 4. Substituting alanines for Ser218 and Ser222 in {Delta}N3, F53S or Y130C abrogated the activation of ERK in the presence of WT B-RAF. Various forms of MEK1 expression vectors were transfected into HEK293 cells with an Myc epitope-tagged WT B-RAF DNA plasmid. Cells were serum-starved for 24 h in medium containing 0.5% FBS and then harvested. Western blot analysis was performed using antibodies against Myc for B-RAF, HA for MEK1, P-ERK and total ERK.

 


Figure 5
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  		Figure 5. The phenylalanine 57 residue of MEK2 is a unique site that harbors gain-of-function mutations. (A) F57S and F57C MEK2 could activate ERK/MAPK without prior upstream signaling from growth factors. Identical amino acid changes were introduced into each counterpart enzyme at equivalent positions. This generated F57S MEK2, F53C MEK1 and Y134C MEK2. The abilities of these variants on endogenous ERK1/2 activation were compared. (B) F53C MEK1 and Y134C MEK2 as well as F53S and Y130C MEK1 require RAF signaling to activate ERK/MAPK. Various MEK1/2 expression vectors were independently transfected into HEK293 cells with (+, B) or without (A) a Myc epitope-tagged WT B-RAF DNA plasmid. Cells were serum-starved for 24 h in medium containing 0.5% FBS. Western blot analysis was performed using antibodies against HA for MEK1 or MEK2, Myc for B-RAF, P-ERK and total ERK. Asterisks denote MEK mutants found in CFC patients.

 
Thus, at least one other MEK mutant can be constitutively active when identical mutations are introduced in the equivalent positions of MEK2. We concluded that the phenylalanine 57 of MEK2 is a unique site that harbors gain-of-function mutations.

F53C MEK1 and Y134C MEK2 mutants require RAF signals to activate ERK1/2
We next tested whether F53C MEK1 and Y134C MEK2 can activate ERK/MAPK after upstream signaling. We transfected various forms of MEK1/2 expression vectors into HEK293 cells together with WT B-RAF DNA plasmid. Cells were serum-starved for 24 h prior to western blotting (Fig. 5B). The effect of WT B-RAF was dramatic with F53C MEK1 (lane 10) and Y134C MEK2 (lane 13). They activated ERK1/2 in a manner similar to F53S (lane 8) and Y130C (lane 12). Thus, F53C MEK1 and Y134C MEK2 require RAF signaling to activate ERK/MAPK. These results also suggest that CFC mutations in phenylalanine 53 or tyrosine 130 of MEK1 may have selective sensitivity to RAF inhibition.

F57C MEK2 is resistant to the RAF inhibitor SB-590885
F57C MEK2 can activate ERK1/2 in the absence of growth factors and without added RAF in cultured cells (Figs 1B, 3B and 5B), suggesting that it might be resistant to pharmacological inhibition of RAF. We therefore tested whether F57C MEK2-stimulated activation of the MAPK signaling cascade could occur in spite of RAF kinase inhibition. We used SB-590885, which selectively inhibits RAF kinases and is more effective against B-RAF than C-RAF (18). Overexpression of F57C MEK2 in HEK293 cells caused ERK phosphorylation (Fig. 6, lanes 5 and 7). Phosphorylation occurred without added B-RAF (lane 5). Treatment with SB-590885 for 60 min did not inhibit F57C MEK2-induced ERK activation (lanes 6 and 8), although we observed that SB-590885 dramatically reduced WT B-RAF-induced MEK2 phosphorylation in the same sample (lane 8).


Figure 6
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  		Figure 6. F57C MEK2 is resistant to the selective RAF inhibitor SB-590885. (A) F57C MEK2 is resistant to SB-590885. About 1.5 µg of F57C and Y134C MEK2 expression vectors were independently transfected into HEK293 cells with (+) or without (–) 0.5 µg of an Myc epitope-tagged DNA plasmid carrying WT B-RAF. The cells were serum-starved for 24 h in medium containing 0.5% FBS and then exposed to 10 µM SB-590885 (+) or DMSO (–) for 60 min prior to harvesting. Western blot analysis was performed using antibodies against Myc for B-RAF, HA for MEK2, S222/S226 P-MEK2, P-ERK and total ERK.

 
Alternatively, Y134C MEK2 stimulated ERK phosphorylation in the presence of WT B-RAF when it was phosphorylated at Ser222 and Ser226 (lane 11). This phenotype was identical to that of the MEK1 mutants F53S and Y130C (Fig. 3A, lanes 12 and 14). SB-590885 blocked phosphorylation of MEK2 and activation of ERK1/2 in the sample transfected with WT B-RAF and Y134C MEK2 (Fig. 6, lane 12).

Phosphorylation of Ser222 and Ser226 is essential for activating F57C MEK2 in the MAPK signaling cascade
The degree to which F57C MEK2 requires phosphorylation of Ser222 and Ser226 to activate ERK/MAPK was not clear. To address this question, we examined the activity of the F57C mutant when Ser222 and Ser226 are substituted to alanines, glutamines or completely deleted (F57C/S222A+S226A, F57C/S222Q+S226Q, F57C/{Delta}S222+{Delta}S226; Fig. 7A, lanes 7–9). We also introduced these mutations into WT MEK2 (S222A+S226A, S222Q+S226Q, {Delta}S222+{Delta}S226; Fig. 7A, lanes 3–5) and then compared activity on endogenous ERK1/2 activation. In F57C, all the mutations dramatically reduced its ability to activate ERK. Moreover, ERK phosphorylation did not even occur in the presence of WT B-RAF (Fig. 7B, lanes 7–9). Thus, phosphorylation of these serines appears to be essential for activating F57C in the MAPK signaling cascade. Results were similar for WT MEK2 (Fig. 7A, lanes 3–5).


Figure 7
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  		Figure 7. Phosphorylation of Ser222 and Ser226 by RAF is essential for activating F57C MEK2 in the MAPK signaling cascade. (A) Replacing Ser222 and Ser226 of F57C MEK2 with alanines (S222A+S226A, lane 7) or glutamines (S222Q+S226Q, lane 8) or deleting these residues ({Delta}S222+{Delta}S226, lane 9) dramatically reduced F57C MEK2's ability to activate ERK, even in the presence of B-RAF. (B) Various MEK1/2 expression vectors were independently transfected into HEK293 cells with (B) or without (A) an Myc epitope-tagged WT B-RAF DNA plasmid. Cells were serum-starved for 24 h in medium containing 0.5% FBS and harvested. Western blot analysis was performed using antibodies against HA for MEK2, Myc for WT B-RAF, P-ERK and total ERK.

 
We conclude that F57C must be activated by RAF for ERK induction. The requirement of RAF signals could, however, be reduced when compared with {Delta}N3, F53S and Y130C MEK1 and Y134C MEK2 mutants. This may be due to an increase in the sensitivity of phosphorylation at Ser222 and Ser226 of the protein to RAF signaling (see P-MEK2 blot in Fig. 6, lanes 7 and 11).

Activation of the MAPK signaling cascade caused by {Delta}N3, F53S or Y130C MEK1, or F57C MEK2 is blocked by the MEK inhibitor U0126
MEK inhibitors can inhibit cancer cell growth and some are undergoing clinical evaluation in that area (3,19). One of these inhibitors is U0126 (20). Because MEK mutants that cause CFC are novel, it is not known whether these CFC mutants are sensitive to MEK inhibition. Therefore, we tested its ability to block MEK mutant-stimulated activation of the MAPK signaling cascade (Fig. 8A and B). U0126 is selective for MEK1 and MEK2 and is non-competitive for ATP and ERK/MAPK (21). Overexpression of {Delta}N3, S218D+S222D, F53S or Y130C MEK1 or S222D+S226D or F57C MEK2 in HEK293 cells increased ERK phosphorylation, as described earlier. U0126 treatment did not inhibit ERK/MAPK activation by S218D+S222D MEK1 or S222D+S226D MEK2. This observation is consistent with a previous finding using PD 184352 another MEK inhibitor (22).


Figure 8
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  		Figure 8. U0126 inhibits activation of the MAPK signaling cascade by {Delta}N3, F53S or Y130C MEK1 or F57C MEK2, but not through S218D+S222D MEK1 or S222D+S226D MEK2. Various MEK1/2 expression vectors were independently transfected into HEK293 cells with (+, A) or without (B) an Myc epitope-tagged DNA plasmid carrying WT B-RAF. The cells were serum-starved for 24 h in medium containing 0.5% FBS and then treated with 50 µM U0126 (+) or DMSO (–) for 30 min prior to harvesting. Western blot analysis was performed using antibodies against HA for MEK1 or MEK2, Myc for B-RAF, P-ERK and total ERK.

 
In contrast, with all the other mutants, U0126 reduced levels of phosphorylated MAPK. These observations suggest that (i) S218D+S222D MEK1 and S222D+S226D MEK2 are insensitive to MEK inhibitors, and (ii) U0126 can inhibit MAPK signaling cascade activation caused by {Delta}N3, F53S or Y130C MEK1 or F57C MEK2. These data indicate that small molecule inhibitors of MEK could be of therapeutic use in CFC.

MAPK docking site of MEK can be an alternative molecular target to selectively inhibit the MEK variants
ERK/MAPK requires a kinase docking site (or D-domain) on its substrate to increase phosphorylation efficiency (23). D-domains have been found in various ERK substrates such as Elk-1, Sap-1, Sap-2, Ets-1, c-Myc and cyclin D1 (24). ERK is a substrate of MEK. In contrast, MEK is unlikely to be a substrate of ERK. In spite of this fact, D-domains are found in the N-terminal amino acids 3–17 and 5–19 of MEK1 and MEK2, respectively (Motif Scan; http://scansite.mit.edu). This suggests that strict recognition of ERK by MEK1/2 is crucial for effective and accurate signal transmission in the cascade. In accordance with this idea, deleting D-domains inhibits MEK1 and MEK2 interactions with ERK and reduces ERK/MAPK activities (25).

To test whether the MEK2 mutant F57C or S222D+S226D binds to MAPK through the D-domain to activate ERK1/2, we completely deleted the D-domain of MEK2 (amino acids 5–19) from WT, F57C or S222D+S226D MEK2 and generated {Delta}D, F57C/{Delta}D and S222D+S226D/{Delta}D MEK2 variants, respectively. We subsequently performed immunoprecipitation (IP) and immunoblotting (IB), following ectopic expression of Flag-tagged ERK2 with either empty vector (lane 1), HA-tagged WT (Fig. 9A, lanes 2 and 3), {Delta}D (lane 4), F57C (lane 5), F57C/{Delta}D (lane 6), S222D+S226D (lane 7) or S222D+S226D/{Delta}D MEK2 (lane 8) in HEK293 cells. ERK2 associated with WT, F57C and S222D+S226D MEK2 (lanes 3, 5 and 7). This association was prevented by deleting the MEK2 D-domain ({Delta}D, lanes 4, 6 and 8). To establish the importance of MEK2–ERK interaction on ERK/MAPK induction in the cultured cells, we examined the activity of the F57C and S222D+S226D MEK2 mutants when the D-domain is completely deleted ({Delta}D). We compared the abilities of these variants on endogenous ERK1/2 activation with other mutants in which the ATP-binding sites of lysines are impaired (K101M), or Ser222 and Ser226 are substituted to alanines (S222A+S226A). Various forms of MEK2 expression vectors were transfected into HEK293 cells and then cells were serum-starved for 24 h prior to harvesting and western blotting (Fig. 9B). Phosphorylation of ERK was significantly reduced by depleting the D-domains (lanes 7 and 11), by impairing the ATP-binding sites of lysines (lanes 8 and 12) or by substituting alanines for serines (lanes 9 and 5) in F57C and S222D+S226D MEK2. These observations suggested that the D-domains, the catalytic sites or key serine residues of MEK1/2 are alternative molecular targets for developing drugs to selectively inhibit CFC MEK variants.


Figure 9
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  		Figure 9. The MAPK docking site (D-domain) of MEK2 is an alternative molecular target for MEK inhibition. (A) Association of ERK2 with WT, F57C and S222D+S226D MEK2 was prevented by deleting the MEK2 D-domain ({Delta}D, lanes 4, 6 and 8). Left side: IP and IB. pcDNA3.1 (lane 1) or various HA-tagged MEK2 plasmids (other lanes) were transfected into HEK 293 cells together with pcDNA3.1 (lane 2) or a Flag-tagged ERK2 expression vector (other lanes). Cells were harvested 24 h later. Samples were precipitated with a Flag epitope tag antibody. Immunoprecipitates were subjected to SDS–PAGE and stained with Flag (ERK2) and HA (MEK2) antibodies. Right: IB analysis with 2% of total cell lysates was a control. (B) Deleting the MEK2 D-domain ({Delta}D, lanes 7 and 11), impairing the ATP site (K101M, lanes 8 and 12) or substituting alanines for serines (S222A+S226A, lanes 9 and 5) abrogated or significantly reduced the abilities of F57C and S222D+S226D MEK2 mutants to activate ERK1/2 (lanes 6 and 10). Various MEK2 expression vectors were independently transfected into HEK293 cells. Cells were serum-starved with medium containing 0.5% FBS for 24 h prior to harvesting. Western blot analysis was performed using antibodies against HA for MEK2, P-ERK and total ERK.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
The Ras/MAPK pathway has been studied intensively in cancer (1–3). Constitutive activation of ERK1/2 is frequently found in human cancer cells derived from a variety of tissues such as pancreas, colon, lung, ovary and kidney (26). Mutations in the upstream components of the MAPK pathway, including K-RAS, N-RAS, H-RAS, C-RAF and B-RAF, have been described and are known to alter signaling in tumors (3). However, prior to our recent findings, no somatic or constitutional mutations had been described in MEK genes (7). Understanding the functions of these novel mutations may be important for understanding MEK's role in the MAPK signaling pathway not only in CFC and cancer, but also as a prospective therapy because these pathways are potential therapeutic targets for these patients. Because CFC has an evolving phenotype, we believe that systemically treating it by inhibiting the Ras/MAPK pathway may merit investigation. Furthermore, this approach may also be useful for developing therapies for the malignancies in this population (9–12). Small molecule therapy targeting the MAPK cascade is in clinical trials for cancer (2,3). The goal of this study was to characterize the novel CFC MEK mutants biochemically and to examine their sensitivities to small molecule inhibitors.

Eight MEK homologs have been identified to date, but only MEK1 and MEK2 are known participants in the Ras/MAPK signaling cascade (1,3,27,28). They are susceptible to phosphorylation by RAF. MEK1 is ubiquitously expressed. It is usually expressed at higher levels than MEK2, although MEK2 predominates in some tissues (29,30). MEK1 gene manipulation in mice causes embryonic death at around 10.5 days of gestation because of remarkably underdeveloped placenta rather than a major anomaly in the embryo (27). In contrast, MEK2-deficient mice are viable and their overall behavior, fertility, body weight and life spans are normal (28). These differences suggest that MEK1 may compensate for MEK2 deficiency, whereas MEK2 cannot. This fact indicates that selective inhibition of MEK2 in CFC patients with MEK2 mutations may be a therapeutic option with relatively low cytotoxicity.

A key role for MEK in tumor development has also been described. MEK activation through the MAPK signaling cascade is necessary for mammalian cell transformation, and constitutively active MEK mutants promote transformation of fibroblast cells (13,31). Furthermore, the MEK inhibitor PD 184352 (CI-1040) can inhibit growth of human and murine colon carcinomas in nude mice (32). We studied the mechanism of this inhibition and found that treating colon carcinoma cells with small molecule MEK inhibitors blocked both CDK4 and CDK2 kinase activities and caused G1 growth arrest (19).

MEK phosphorylates ERK1/2 on both a tyrosine and a threonine residue, rendering it active (1,3). MEK1 and MEK2 themselves are phosphorylated and activated by RAF in two key serine residues in the regulatory loop, Ser218/Ser222 in MEK1 and Ser222/Ser226 in MEK2. Replacing these serine residues with negatively charged amino acids such as aspartate (S218D+S222D MEK1 or S222D+S226D MEK2) may mimic the modification made by phosphorylation and results in a constitutively active kinase whose MEK activity is 85x greater than the WT enzyme's (13). Another regulatory area in MEK is an N-terminal region of {alpha}-helix (amino acids 32–51 of MEK1) located outside its kinase catalytic core. This area has been characterized as an auto-inactivation domain, and deleting it ({Delta}N3 MEK1) renders MEK 45x more active than WT enzyme (13).

In this article, we studied the biochemical functions of CFC MEK mutants such as F53S and Y130C MEK1 and F57C MEK2. In addition, we recently identified Y134C MEK2 as causative for CFC (Rauen, unpublished data). F53S MEK1 and F57C MEK2 are of particular interest because we identified missense mutations in these genes outside the protein kinase core domain (7). This finding suggested that the N-terminal region of MEK might have an important regulatory role, in addition to its role in substrate recognition (22). In this study, we compared endogenous ERK1/2 activation in these mutants with other artificially generated constitutively active MEK mutants such as {Delta}N3 and S218D+S222D MEK1 and S222D+S226D MEK2.

In this work, we overexpressed the novel CFC MEK mutants in the cultured cells simply as a way to facilitate analysis. Our choice of this approach was not intended to imply that the mutants are overexpressed in CFC. Another system to characterize the biochemical function of the mutants and their sensitivities to small molecule inhibitors could be performed in primary fibroblast or other cells from CFC patients. Unfortunately, CFC is an extremely rare condition, which limits the cell donor pool. In addition, the only CFC cells currently available are Epstein–Barr virus (EBV)-transformed lymphoblast cells. EBV infection alters cellular signaling, including the MAPK pathway, making their utility in this context extremely limited. Thus, and to re-iterate, given the current situation, we believe that using overexpressed proteins is the most practical approach for accomplishing our purpose.

Our study shows that the MEK variants found in CFC must be activated by RAF to induce ERK. However, F57C MEK2's RAF requirement is much less than that of mutants such as F53S and Y130C MEK1 and Y134C MEK2. This difference made F57C MEK2 resistant to the selective RAF inhibitor SB-590885 in cultured cells. However, all the CFC mutants were sensitive to the MEK inhibitor U0126 in the same cells. CFC patients mostly have mutations in B-RAF (7,8), and a recent report indicates the possibility that cancer cells with activated B-RAF have enhanced, selective sensitivity to MEK inhibitors (33). Thus, regardless of mutations in an individual with CFC, MEK inhibition may be a potential therapeutic approach.

PD 184352 (CI-1040) and U0126 are widely used MEK inhibitors. They are highly selective and bind to a hydrophobic pocket in a region without sequence homology to other kinases (3,17). These compounds do not block the activities of S218D+S222D MEK1 and S222D+S226D MEK2 (this study, 22). However, in this study, deleting the D-domain of MEK2 dramatically reduced activities of S222D+S226D and F57C MEK2. Thus, the D-domains of MEK1/2 are alternative molecular targets for selectively inhibiting the MEK variants studied here.

The significance of targeting the MEK1/2 D-domain in malignancies has been demonstrated in a study of the anthrax toxin's lethal factor (LF) (34,35). LF is a zinc metalloprotease that inactivates MEK1/2 through enzymatic cleavage in its N-terminus (34). LF also cleaves the N-terminus of MKK3b, MKK4, MKK6 and MKK7 (36,37). This action causes a severe reduction of ERK1/2 and p38 MAPK activities, resulting in inhibition of RAS V12-mediated transformation in vitro and in vivo (35). Interestingly, the cleaved N-terminus contains the D-domain of each molecule. These observations support continued investigation into targeting MEK D-domains in CFC, although further studies will be necessary to develop a clinical application for both CFC and cancer treatment.

We have proposed that small molecule inhibitors of MEK could be of therapeutic use in CFC. Because the activities of the mutants may indeed influence development in the absence of overexpression, inhibiting them can still alter the course of disease in CFC patients. We believe that this first attempt may advance an idea of a potential MAPK inhibitor therapy for CFC patients to a next step. MAPK activities are likely to be activated with other Ras/MAPK genetic syndromes such as CS. We consider that MEK inhibition is a potential therapeutic approach for CS as well.


   MATERIALS AND METHODS
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
FUNDING
 REFERENCES
 
Chemical, cell line and transfection
U0126 (Promega, Madison, WI, USA) and SB590885 (Calbiochem, San Diego, CA, USA) were suspended in DMSO. Human recombinant EGF was purchased from Promega Corporation. HEK 293 cells and African green monkey kidney fibroblast-like (COS-7) cells were obtained from the American Type Culture Collection. Cells were cultured with RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) and penicillin and streptomycin. They were maintained in a 5% CO2 incubator at 37°C. FuGENE6 (Roche, Indianapolis, IN, USA) was used for transient transfection.

DNA plasmids and site-directed mutagenesis
Hemagglutinin (HA) epitope-tagged WT rat MEK1 and MEK2 cDNAs (Upstate, Charlottesville, VA, USA) were subcloned into a pcDNA3.1 expression vector (Invitrogen). MEK mutants were generated with a QuickChange or ExSite site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA), according to the manufacturer's instructions. The Myc epitope-tagged WT B-RAF pcDNA3.1 expression vector has been described (7).

Western blot analysis
Total cellular protein was prepared using cell lysis buffer (Cell Signaling, Danvers, MA, USA). Equal amounts of total protein were resolved by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE). Western blots were developed by enhanced chemiluminescence (Upstate). The following primary and secondary antibodies were used: phospho-MEK1/2 (9121, Cell Signaling), phospho-ERK1/2 (E-4, Santa Cruz, CA, USA), total (p42 and p44) ERK (Promega), Myc (A-14, Santa Cruz), HA horseradish peroxidase (HRP)-conjugated (12CA5, Roche) and Sheep anti-mouse IgG HRP and Donkey anti-rabbit IgG HRP (Amersham, Piscataway, NJ, USA).

IP and IB analysis
Various forms of HA-tagged rat MEK2 DNA plasmids were transfected into HEK293 cells together with a Flag-tagged human ERK2 expression vector (24). Cells were collected 24 h later. Samples were precipitated with a Flag epitope tag antibody (M2 Agarose-conjugated, Sigma, St Louis, MO, USA). Immunoprecipitates were subjected to SDS–PAGE and subsequently stained with HA (12CA5 HRP-conjugated, Roche) or Flag (M2 HRP-conjugated, Sigma) antibody.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
REFERENCES
 
This work was supported by grants from the Concern Foundation, Daiichi–Sankyo Pharmaceuticals, the Simon E. Guzik Memorial Foundation, the UCSF Research-Evaluation Allocation Committee, the V Foundation to O.T. and the Wood Foundation to F.M. K.A.R. was supported by the NIH grant HD048502.


   ACKNOWLEDGEMENTS
 
We are grateful to Drs Masashi Aonuma and Valerie Natale for their helpful comments.

Conflict of Interest statement. None of authors has any conflicts of interest.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 

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